Preparation method of tremella gum

Abstract

This invention belongs to the field of daily chemical products technology, specifically relating to a method for preparing Tremella fuciformis gel, comprising the following steps: (1) pulverizing dried Tremella fuciformis fruiting bodies, sieving to obtain Tremella fuciformis powder; (2) mixing Tremella fuciformis powder and extraction solvent and then ultrasonically treating to obtain a mixture; (3) adding inorganic alkali to the mixture and stirring, while slowly adding propylene oxide dropwise, maintaining the reaction temperature, adding an acid to adjust the pH to 5-8, and drying to obtain the Tremella fuciformis gel; wherein, the extraction solvent comprises betaine, polyethylene glycol and lactic acid in a molar ratio of 1:(2-4):(0.4-0.8). The Tremella fuciformis gel prepared by this invention has high viscosity and good light transmittance under normal temperature conditions and at low concentrations, enabling the prepared Tremella fuciformis gel to be used as a thickener in daily chemical products. Moreover, the Tremella fuciformis gel prepared by this invention also has good antioxidant and whitening effects, broadening the application range of the Tremella fuciformis gel prepared by this invention.

Description

Technical Field

[0001] This invention belongs to the field of daily chemical products technology, specifically relating to a method for preparing tremella gel. Background Technology

[0002] Tremella, also known as white fungus, is an edible fungus that is both food and medicine. It is rich in polysaccharides, amino acids, proteins and various minerals. It has functions such as anti-oxidation, anti-aging, anti-tumor, blood pressure reduction and immune enhancement. These functional properties have led to the widespread application of tremella polysaccharides extracted from tremella in many fields such as medicine, health care, food processing, skin care and beauty, and animal husbandry.

[0003] The polysaccharides contained in the fruiting body of Tremella fuciformis include intracellular polysaccharides, extracellular polysaccharides, and cell wall polysaccharides. Because these polysaccharides are distributed throughout different cellular structures of the fruiting body, traditional processing methods often result in low extraction efficiency of active ingredients or damage to other active components. For example, processing with high-concentration ethanol solutions can cause protein denaturation in Tremella fuciformis, leading to a decline in the quality of the final product.

[0004] In conclusion, there is an urgent need to develop a method for preparing tremella gel that can fully extract the effective components from tremella and reduce raw material loss, so as to meet the high market demand for tremella gel. Summary of the Invention

[0005] In view of this, a tremella glue and its preparation method are proposed to solve the above problems.

[0006] In a first aspect, the present invention provides a method for preparing Tremella fuciformis gelatin, comprising the following steps:

[0007] (1) The dried fruiting body of Tremella fuciformis is crushed and sieved to obtain Tremella fuciformis powder;

[0008] (2) The mixture of tremella powder and extraction solvent is subjected to ultrasonic treatment to obtain a mixture;

[0009] (3) Add an inorganic base to the mixture and stir to mix. While stirring, slowly add propylene oxide, keep the reaction at a constant temperature, add an acid to adjust the pH to 5-8, and dry to obtain the tremella gel.

[0010] The extraction solvent comprises betaine, polyethylene glycol, and lactic acid in a molar ratio of 1:(2-4):(0.4-0.8).

[0011] This invention incorporates betaine, polyethylene glycol, and lactic acid into the extraction solvent, which effectively disrupts the cell structure of *Tremella fuciformis* fruiting bodies, thereby releasing the active ingredients. Specifically, betaine acts as a hydrogen bond acceptor, while polyethylene glycol and lactic acid act as hydrogen bond donors. Together, they form a hydrogen bond network within the eutectic solvent, effectively disrupting the hydrogen bonds or van der Waals forces in the *Tremella fuciformis* fruiting body cell structure, thus releasing the active ingredients. Furthermore, by using polyethylene glycol and lactic acid as hydrogen bond donors, this invention can also adjust the polarity and pH value of the eutectic solvent to better penetrate the *Tremella fuciformis* fruiting body cell structure, thereby facilitating the better dissolution of the active ingredients.

[0012] Furthermore, in step (2), the water content of the extraction solvent is 15wt%-20wt%.

[0013] The water content of the extraction solvent in this invention is 15wt%-20wt%. The reasonable water content in the extraction solvent can improve the viscosity and flowability of the extraction solvent, thereby achieving the purpose of dissolving the active ingredients in the fruiting body of Tremella fuciformis from the cell structure of the fruiting body.

[0014] Further, in step (2), the ratio of the tremella powder to the extraction solvent is 1g:5-10mL.

[0015] The material-to-liquid ratio of the Tremella fuciformis powder to the extraction solvent in this invention is 1g:5-10mL. This reasonable material-to-liquid ratio not only ensures that the Tremella fuciformis powder and the extraction solvent are in full contact, promoting the dissolution of the active ingredients in the Tremella fuciformis fruiting body into the extraction solvent, but also avoids inconsistent dissolution of intracellular polysaccharides, extracellular polysaccharides and cell wall polysaccharides, which would lead to their conversion into reducing sugars in the extraction solvent, resulting in a decrease in the viscosity and performance of the finished product.

[0016] Furthermore, the ultrasonic treatment is performed for 13-17 minutes at a temperature of 40-80℃ and an ultrasonic frequency of 100-300W.

[0017] Furthermore, the ultrasonic treatment involves first ultrasonicating for 10-12 minutes at a temperature of 40-50℃ and an ultrasonic frequency of 100-180W, and then ultrasonicating for 3-5 minutes at a temperature of 70-80℃ and an ultrasonic frequency of 200-300W.

[0018] In this invention, by controlling the ultrasonic temperature, ultrasonic power, and ultrasonic time, not only can the extraction solvent better penetrate into the cell structure of the Tremella fruiting body and fully destroy the cell structure, thereby improving the extraction efficiency of active ingredients in the Tremella fruiting body, but it can also avoid protein denaturation in the Tremella fruiting body, which would lead to blockage of the cell structure and thus reduce the dissolution efficiency of active ingredients in the Tremella fruiting body.

[0019] Furthermore, based on the mass of the tremella powder, the amount of propylene oxide used is 30%-50%.

[0020] Furthermore, based on the mass of the tremella powder, the amount of the inorganic alkali used is 10% to 25%.

[0021] Further, in step (3), the inorganic base is any one of sodium bicarbonate, sodium carbonate, sodium hydroxide, potassium hydroxide, potassium carbonate, and potassium bicarbonate.

[0022] Furthermore, the acid is any one of hydrochloric acid, citric acid, glacial acetic acid, tannic acid, and trichloroacetic acid.

[0023] Further, in step (3), the stirring speed is 200-400 r / min, the temperature is 45-55℃, and the time is 0.25-0.5 h; the stirring speed when adding propylene oxide is 200-300 r / min, the temperature is 50-70℃, and the time is 1-2 h; and the stirring speed when maintaining the temperature is 50-150 r / min, the temperature is 50-70℃, and the time is 2-4 h.

[0024] In this invention, propylene oxide is used to treat the pretreated tremella powder, which can further improve the viscosity and transparency of the finished product.

[0025] Furthermore, the polyethylene glycol is polyethylene glycol 400.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] This invention first uses a eutectic solvent to ultrasonically treat the fruiting bodies of *Tremella fuciformis*, then adds propylene oxide dropwise under alkaline conditions and stirs the reaction. The resulting *Tremella fuciformis* glue exhibits high viscosity and good light transmittance at room temperature and low concentrations, making it suitable as a thickener in daily chemical products. More importantly, the *Tremella fuciformis* glue prepared by this invention also possesses excellent antioxidant and whitening effects, broadening the application range of the glue.

[0028] This invention incorporates betaine, polyethylene glycol, and lactic acid into the extraction solvent. By controlling parameters such as the water content, solid-liquid ratio, and ultrasonic processing in the extraction solvent, the cell structure of the Tremella fruiting body can be thoroughly disrupted, thereby releasing the active ingredients from the cell structure. More importantly, the interaction between betaine, polyethylene glycol, lactic acid, and Tremella gum in this invention further enhances the moisturizing properties of the finished product. Attached Figure Description

[0029] Figure 1 Ion chromatogram of Tremella fuciformis gel in Example 3 of this invention

[0030] Figure 2 Gel permeation chromatogram of Tremella fuciformis gel in Example 3 of this invention

[0031] Figure 3 Schematic diagram of the hair removal area of ​​the experimental subject in the skin phototoxicity test of this invention.

[0032] Figure 4 Results of multiple skin irritation tests on Tremella fuciformis gel in Example 3 of this invention

[0033] Figure 5 Results of the acute eye irritation test of Tremella fuciformis gel in Example 3 of this invention

[0034] Figure 6 Example 3 of the present invention: Skin allergy test results of tremella gelatin

[0035] Figure 7 Example 3 of the present invention: Skin phototoxicity test results of tremella gelatin Detailed Implementation

[0036] This invention provides a Tremella fuciformis gel, its preparation method, and its application. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0037] Unless otherwise specified, the experimental methods used in the examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0038] Example 1

[0039] A method for preparing tremella gelatin includes the following steps:

[0040] (1) Pulverize the dried fruiting body of Tremella fuciformis and pass it through a 100-mesh sieve to obtain Tremella fuciformis powder;

[0041] (2) Mix the tremella powder and the extraction solvent at a ratio of 1g:5mL. First, sonicate at 40℃ and 100W for 12min, then sonicate at 70℃ and 200W for 5min to obtain the mixture.

[0042] (3) Add sodium hydroxide to the mixture and stir to mix. While stirring, slowly add propylene oxide dropwise, keep the reaction at a constant temperature, add hydrochloric acid to adjust the pH to 5, and dry to obtain the finished product.

[0043] In step (2), the extraction solvent is prepared by mixing betaine, polyethylene glycol 400 and lactic acid in a molar ratio of 1:2:0.4, adding deionized water and mixing well to obtain an extraction solvent with a water content of 15wt%.

[0044] In step (3), based on the mass of the tremella powder, the amount of sodium hydroxide is 10% and the amount of propylene oxide is 30%; the stirring speed is 200 r / min, the temperature is 45℃, and the time is 0.5 h; the stirring speed when adding propylene oxide is 200 r / min, the temperature is 50℃, and the time is 2 h; the stirring speed when keeping warm is 50 r / min, the temperature is 50℃, and the time is 4 h.

[0045] Example 2

[0046] A method for preparing tremella gelatin includes the following steps:

[0047] (1) Pulverize the dried fruiting body of Tremella fuciformis and pass it through a 100-mesh sieve to obtain Tremella fuciformis powder;

[0048] (2) Mix the tremella powder and the extraction solvent at a ratio of 1g:10mL. First, sonicate at 50℃ and 180W for 10min, then sonicate at 80℃ and 300W for 3min to obtain the mixture.

[0049] (3) Add sodium hydroxide to the mixture and stir to mix. While stirring, slowly add propylene oxide dropwise, keep the reaction at a constant temperature, add hydrochloric acid to adjust the pH to 8, and dry to obtain the finished product.

[0050] In step (2), the extraction solvent is prepared by mixing betaine, polyethylene glycol 400 and lactic acid in a molar ratio of 1:4:0.8, adding deionized water and mixing well to obtain an extraction solvent with a water content of 20wt%.

[0051] In step (3), based on the mass of the tremella powder, the amount of sodium hydroxide is 25% and the amount of propylene oxide is 50%; the stirring speed is 400 r / min, the temperature is 55℃, and the time is 0.25 h; the stirring speed when adding propylene oxide is 300 r / min, the temperature is 70℃, and the time is 1 h; the stirring speed for the heat preservation reaction is 150 r / min, the temperature is 70℃, and the time is 2 h.

[0052] Example 3

[0053] A method for preparing tremella gelatin includes the following steps:

[0054] (1) Pulverize the dried fruiting body of Tremella fuciformis and pass it through a 100-mesh sieve to obtain Tremella fuciformis powder;

[0055] (2) Mix the tremella powder and the extraction solvent at a ratio of 1g:8mL. First, sonicate at 45℃ and 150W for 11min, then sonicate at 75℃ and 250W for 4min to obtain the mixture.

[0056] (3) Add sodium hydroxide to the mixture and stir to mix. While stirring, slowly add propylene oxide dropwise, keep the reaction at a constant temperature, add hydrochloric acid to adjust the pH to 7, and dry to obtain the finished product.

[0057] In step (2), the extraction solvent is prepared by mixing betaine, polyethylene glycol 400 and lactic acid in a molar ratio of 1:3:0.5, adding deionized water and mixing well to obtain an extraction solvent with a water content of 18wt%.

[0058] In step (3), based on the mass of the tremella powder, the amount of sodium hydroxide is 15% and the amount of propylene oxide is 40%; the stirring speed is 300 r / min, the temperature is 50℃, and the time is 0.4 h; the stirring speed when adding propylene oxide is 250 r / min, the temperature is 60℃, and the time is 1.5 h; the stirring speed when maintaining the temperature is 100 r / min, the temperature is 60℃, and the time is 3 h.

[0059] Comparative Example 1

[0060] Compared with Example 3, the only difference is the ratio of raw materials used in the extraction solvent. The extraction solvent in Comparative Example 1 was prepared by mixing betaine, polyethylene glycol 400 and lactic acid in a molar ratio of 1:0.5:3, adding deionized water and mixing well to obtain an extraction solvent with a water content of 18wt%. The remaining steps and parameters were the same as in Example 3.

[0061] Comparative Example 2

[0062] Compared with Example 3, the only difference is the ratio of raw materials used in the extraction solvent. The extraction solvent in Comparative Example 2 was prepared by mixing betaine, polyethylene glycol 400 and lactic acid in a molar ratio of 2:3:0.5, adding deionized water and mixing well to obtain an extraction solvent with a water content of 18wt%. The remaining steps and parameters were the same as in Example 3.

[0063] Comparative Example 3

[0064] Compared with Example 3, the only difference is the type of raw material for the extraction solvent, namely, citric acid is used instead of lactic acid. The other steps and parameters are the same as in Example 3.

[0065] Comparative Example 4

[0066] Compared with Example 3, the only difference is the type of raw material for the extraction solvent, that is, polyethylene glycol 600 is used instead of polyethylene glycol 400. The other steps and parameters are the same as in Example 3.

[0067] Comparative Example 5

[0068] Compared with Example 3, the only difference is the material-to-liquid ratio of the tremella powder and the extraction solvent. The tremella powder and the extraction solvent are mixed at a material-to-liquid ratio of 1g:3mL. The remaining steps and parameters are the same as in Example 3.

[0069] Comparative Example 6

[0070] Compared with Example 3, the only difference is the material-to-liquid ratio of the tremella powder and the extraction solvent. The tremella powder and the extraction solvent are mixed at a material-to-liquid ratio of 1g:12mL. The remaining steps and parameters are the same as in Example 3.

[0071] Comparative Example 7

[0072] Compared with Example 3, the only difference is the ultrasonic process parameters. The tremella powder and extraction solvent are mixed at a material-to-liquid ratio of 1g:8mL and ultrasonicated for 15min at a temperature of 45℃ and an ultrasonic frequency of 150W to obtain a mixture. The remaining steps and parameters are the same as in Example 3.

[0073] Comparative Example 8

[0074] Compared with Example 3, the only difference is the ultrasonic process parameters. The tremella powder and extraction solvent are mixed at a material-to-liquid ratio of 1g:8mL and ultrasonicated for 15min at a temperature of 75℃ and an ultrasonic frequency of 250W to obtain a mixture. The remaining steps and parameters are the same as in Example 3.

[0075] Comparative Example 9

[0076] Compared with Example 3, the only difference is the ultrasonic process parameters. First, the ultrasonic process is carried out at a temperature of 45°C and an ultrasonic frequency of 150W for 4 minutes, and then at a temperature of 75°C and an ultrasonic frequency of 250W for 11 minutes. The remaining steps and parameters are the same as in Example 3.

[0077] Experimental Example 1

[0078] The viscosity, light transmittance, moisturizing properties, and antioxidant properties of the tremella glue prepared in Examples 1-3 and Comparative Examples 1-9 were tested.

[0079] The viscosity and transmittance were tested by adding deionized water to the prepared product at 25°C to prepare a test solution with a mass concentration of 1%. The viscosity of the test solution was measured using an NDJ-1 rotary viscometer, and the transmittance was measured at 400 nm using an ultraviolet spectrophotometer.

[0080] Moisturizing performance is demonstrated through moisture absorption rate and moisture retention rate data, and the testing method is as follows:

[0081] Prepare saturated Na2CO3 solutions and place them in desiccators to create a test environment with a relative humidity of 43%.

[0082] Determination of moisture absorption rate: Accurately weigh 0.3g of the tremella fuciformis gelatin products prepared in Examples 1-3 and Comparative Examples 1-9, and place them separately into weighing bottles that have been dried to constant weight. Then place them in a desiccator with a humidity of 43%. Weigh the samples after 5 hours, and calculate the in vitro moisture absorption rate according to the following formula:

[0083] Moisture absorption rate (%) = (M2 - M1) / M1 × 100%

[0084] In the formula, M1 is the mass before being placed in the dryer, and M2 is the mass when it is taken out of the dryer.

[0085] Determination of moisturizing rate: The Tremella fuciformis gel products obtained in Examples 1-3 and Comparative Examples 1-9 were prepared into 1 wt% sample solutions using deionized water. These solutions were then placed in weighing bottles that had been dried to constant weight and placed in a desiccator with a humidity of 43%. The weight of the samples was measured after 12 hours, and the in vitro moisturizing rate was calculated using the following formula:

[0086] Moisturizing rate (%) = M2 / M1 × 100%

[0087] In the formula, M1 is the mass before being placed in the dryer, and M2 is the mass when it is taken out of the dryer.

[0088] Antioxidant performance is demonstrated by hydroxyl radical scavenging rate data, and the test method for hydroxyl radical scavenging rate is as follows:

[0089] The tremella gelatin products obtained in Examples 1-3 and Comparative Examples 1-9 were prepared into test sample solutions with a concentration of 1 wt% using deionized water.

[0090] Salicylic acid was prepared into a 2 mmol / L salicylic acid-ethanol solution using anhydrous ethanol.

[0091] FeSO4·6H2O was prepared into a ferrous sulfate solution with a concentration of 6 mmol / L using deionized water;

[0092] H2O2 was diluted with deionized water to a concentration of 6 mmol / L.

[0093] Sample group: Mix 1 mL of the sample solution to be tested, 1 mL of ferrous sulfate solution, 1 mL of salicylic acid-ethanol solution, and 1 mL of H2O2 solution evenly, and incubate in a water bath at 37℃ in the dark for 30 min. Measure the absorbance at 510 nm, set up three parallel samples, calculate the average value, and record it as A1.

[0094] Control group: Mix 1 mL of the test sample solution, 1 mL of ferrous sulfate solution, 1 mL of salicylic acid-ethanol solution and 1 mL of H2O evenly, and incubate in a water bath at 37℃ in the dark for 30 min. Measure the absorbance at 510 nm, set up three parallels, calculate the average value and record it as A2.

[0095] Blank group: Mix 1 mL of deionized water, 1 mL of ferrous sulfate solution, 1 mL of salicylic acid-ethanol solution, and 1 mL of H2O2 solution evenly, and incubate in a water bath at 37℃ in the dark for 30 min. Measure the absorbance at 510 nm, set up three parallel samples, calculate the average value, and record it as A0.

[0096] The formula for calculating the clearance rate is as follows:

[0097] Clearance rate (%) = [1 - (A1 - A2) / A0] × 100%

[0098] The experimental results for viscosity, light transmittance, moisturizing properties and antioxidant properties are shown in Table 1.

[0099] Table 1

[0100] Group Viscosity / mPa.s transmittance / % Moisture absorption rate / % Moisturizing rate / % Clearance rate (%) Example 1 11394 85.6 15.2 94.3 92.4 Example 2 11432 84.2 14.8 95.1 93.6 Example 3 11527 88.5 16.3 95.7 95.1 Comparative Example 1 10271 54.3 7.5 80.2 79.5 Comparative Example 2 10539 43.8 7.1 77.3 81.3 Comparative Example 3 11247 82.2 13.6 89.2 90.2 Comparative Example 4 11308 82.8 12.8 92.4 91.3 Comparative Example 5 11283 79.6 12.5 90.1 90.8 Comparative Example 6 11214 76.1 11.1 87.6 88.4 Comparative Example 7 11225 70.5 11.8 86.2 86.7 Comparative Example 8 10952 63.7 8.6 82.9 84.6 Comparative Example 9 10914 67.4 10.2 84.4 85.1

[0101] Experimental results show that the tremella glue prepared by this invention not only has high viscosity and good light transmittance, making it suitable as a thickener in daily chemical products, but also has good moisturizing properties. It can replenish skin moisture, help the skin absorb quickly, maintain a certain moisture content in the stratum corneum, delay moisture loss, and increase water penetration between the epidermis and dermis to restore skin luster and elasticity. It also has good antioxidant properties.

[0102] Compared to Example 3, the raw material ratio of the extraction solvent was adjusted in Comparative Examples 1-2, and the types of raw materials were adjusted in Comparative Examples 3-4, resulting in a decrease in the performance of the final product. This may be because betaine, polyethylene glycol, and lactic acid together form a hydrogen bond network in the eutectic solvent, which can effectively disrupt the hydrogen bonds or van der Waals forces in the cell structure of the Tremella fruiting body, thereby releasing the active ingredients in the Tremella fruiting body. Furthermore, the present invention uses polyethylene glycol and lactic acid as hydrogen bond donors, which can also enhance the polarity of the eutectic solvent and adjust its pH value, helping the extraction solvent to better penetrate into the cell structure of the Tremella fruiting body, thereby better dissolving the active ingredients in the Tremella fruiting body.

[0103] Compared to Example 3, adjusting the material-to-liquid ratio between the Tremella fuciformis powder and the extraction solvent in Comparative Examples 5-6 resulted in a decrease in the performance of the final product. This may be because a reasonable material-to-liquid ratio not only ensures sufficient contact between the Tremella fuciformis powder and the extraction solvent, which is beneficial for promoting the dissolution of active ingredients in the Tremella fuciformis fruiting body into the extraction solvent, but also avoids inconsistent dissolution of intracellular polysaccharides, extracellular polysaccharides, and cell wall polysaccharides, which would lead to their conversion into reducing sugars in the extraction solvent, resulting in a decrease in the viscosity of the final product.

[0104] Compared to Example 3, adjusting the ultrasonic treatment process parameters in Comparative Examples 7-9 resulted in a decrease in the performance of the final product. This may be because appropriate ultrasonic treatment process parameters not only allow the extraction solvent to better penetrate the cell structure of the Tremella fruiting body, fully disrupting the cell structure and thus improving the extraction efficiency of active ingredients, but also prevent protein denaturation in the Tremella fruiting body, which alters the cell structure and reduces the dissolution efficiency of active ingredients.

[0105] Experimental Example 2

[0106] The tremella gelatin products obtained in Examples 1-3 were diluted with deionized water to different concentrations, as per the reference.

[0107] The T / SHRH 015-2018 Cosmetics - Tyrosinase Activity Inhibition Test Method was used to conduct the tyrosinase inhibition test. Each finished product was tested in 3 parallel experiments, and the mean ± standard deviation was calculated. The test results are shown in Table 2.

[0108] Table 2

[0109]

[0110] In the biosynthesis of melanin in the skin, tyrosinase is a key enzyme that acts on dopa to form dopaquinone, which then spontaneously undergoes a series of reactions to finally form melanin. The whitening efficacy of the finished product is evaluated by detecting tyrosinase activity.

[0111] Experimental results show that the tremella gel prepared by this invention has a significant inhibitory effect on tyrosinase activity.

[0112] Experimental Example 3

[0113] The transdermal properties of the tremella gel prepared in Examples 1-3 were tested according to GB / T27818-2011 "In vitro test method for skin absorption of chemicals" and using a PYJ-12B drug transdermal diffusion tester. Each product was tested in 3 parallel experiments, and the average value was calculated. The test results are shown in Table 3.

[0114] Experimental procedure:

[0115] 1. Preparation of test samples

[0116] Citric acid was used as a solvent to adjust the pH value of deionized water to 5-6.

[0117] The finished products of Examples 1-3 were prepared into test samples with a polysaccharide content of 6.5 mg / g and a concentration of 1 wt% using solvents.

[0118] 2. Equipment Preparation

[0119] Place the matching magnetic stir bar in the 8mL receiving cell, fix the pigskin sample on the Franz transdermal cell, with the dermis facing the receiving cell containing PBS and the stratum corneum exposed in the drug delivery cavity. Pull the pigskin taut with tweezers and secure it with clamps. Expel all air, ensuring the bottom surface of the pigskin is in close contact with the receiving solution.

[0120] 3. Skin sample addition

[0121] According to 0.125g / cm 2 For the sample loading amount, accurately weigh 0.275g of the test sample onto the fixed pigskin and place it in a constant temperature water bath at 32℃±1℃. Place a stir bar in the receiving tank and stir continuously at a uniform speed of 300r / min for 24h under light-protected conditions.

[0122] 4. Sampling and Sample Preparation

[0123] Subcutaneous receiving fluid sampling: Take 1 mL of subcutaneous receiving fluid from each test group at the corresponding time points (1h, 1.5h, 2h, 4h, 8h, 24h) for testing. After taking it out, add 1 mL of phosphate buffered saline (PBS) with pH 7.2 to the receiving pool of each test group to continue the transdermal test at subsequent time points.

[0124] Table 3

[0125] name Example 1 Example 2 Example 3 <![CDATA[24h polysaccharide accumulation (ug / cm 2 )]]> 15.03 14.85 15.27

[0126] Experimental results show that the tremella gel prepared by this invention has good transdermal effect, can penetrate the skin barrier and reach the bottom layer of the skin to exert antioxidant and whitening effects.

[0127] Test Example 4

[0128] 1. The Tremella fuciformis gel from Example 3 was prepared into a 10 ppm sample solution using deionized water. The solution was vortexed until homogeneous, filtered through a 0.22 μm aqueous filter membrane, and then used for mineral determination using an ICS-6000 ion chromatograph. The results are shown in [Figure 1]. Figure 1 .

[0129] 2. Accurately weigh the Tremella fuciformis gel sample from Example 3, add deionized water to prepare a sample solution with a concentration of 5 mg / mL, vortex to homogenize, filter through a 0.22 μm aqueous filter membrane, transfer the sample solution to a 1.8 mL vial for later use, and determine the molecular weight using gel permeation chromatography. The results are shown in [Figure number missing]. Figure 2 .

[0130] 3. Weigh 10 mg of the standard sample and the finished product of Example 3 precisely with a tin capsule or an aluminum capsule. After sealing them tightly, put each sample into the auto-sampler of the Flashsmart organic element analyzer to start the detection, and determine the nitrogen element content. The nitrogen content in the tremella glue of Example 3 of the present invention is 0.7 wt%.

[0131] See Figure 1 , indicating that the preparation method of the tremella glue of the present invention retains the mineral active ingredients in tremella, enabling it to achieve the purpose of skin repair and anti-aging by enhancing the skin barrier function and promoting epidermal cell differentiation.

[0132] See Figure 2 , the weight-average molecular weight of the tremella glue prepared by the present invention is about 1.3 million. The tremella glue not only contains polysaccharide structures with large molecular weights, but also polysaccharide structure homologues, and there are also many substances with different molecular weights. Therefore, the molecular weight distribution of the tremella glue prepared by the present invention is the widest, which may be because the preparation method of the present invention fully retains all the nutritional components of the tremella glue.

[0133] Test Example 5

[0134] The tremella glue prepared in Example 3 was subjected to multiple skin irritation tests, acute eye irritation tests, skin allergy tests and skin phototoxicity tests in accordance with the "Technical Specifications for Cosmetics Safety" (2015 Edition), Chapter 6 - Toxicological Test Methods. The test results are shown in Figure 4-7 .

[0135] Multiple skin irritation tests:

[0136] 1. Test animals: 4 New Zealand rabbits of ordinary grade (female, 1912.35 - 2010.54 g), provided by the Guangdong Provincial Medical Experimental Animal Center (Sanshui Base), production license number: SCXK (Guangdong) 2023 - 0035; quality certificate number: 44411600017507. Adapt in the experimental animal house for at least 7 days before the test.

[0137] 2. Breeding environment: Ordinary environment, temperature 16°C - 26°C, relative humidity 30% - 70%, use license number: SYXK (Guangdong) 2022 - 0194.

[0138] 3. Feed source: Guangdong Provincial Medical Experimental Animal Center, production license number: Guangdong Feed Certificate (2024) 05073, production date: 2025 - 06 - 04; feed certificate number: NO.2506060043 - 1.

[0139] 4. Test method: 24 hours before the test, shave the hair on both sides of the spine on the back of the test animals without damaging the epidermis. The hair removal area is 3 cm × 3 cm on each side. Take 0.5 mL of the test substance (the finished product of Example 3 diluted with deionized water to a concentration of 1 mg / mL) and apply it to the shaved skin on the left side with an area of 2.5 cm × 2.5 cm. The skin on the other side is used as a control. Apply it once a day for 14 consecutive days. Starting from the second day, shave the hair before each application, remove the residual test substance with warm water, and observe the local skin reaction and score it 1 hour later.

[0140] Acute eye irritation test:

[0141] 1. Test animals: 3 ordinary New Zealand rabbits (female, 1728.49 - 1750.95 g), provided by the Guangdong Provincial Center for Medical Laboratory Animals (Sanshui Base), production license number: SCXK(Yue)2023 - 0035; quality certificate number: 44411600017507. Adapt to the experimental animal room for at least 7 days before the test.

[0142] 2. Breeding environment: Ordinary environment, temperature 16°C - 26°C, relative humidity 30% - 70%, use license number: SYXK(Yue)2022 - 0194.

[0143] 3. Feed source: Guangdong Provincial Center for Medical Laboratory Animals, production license number: Yue Si Zheng (2024) 05073, production date: 2025 - 05 - 06; feed certificate number: NO.2506060043 - 1.

[0144] 4. Test method: Take 0.1 mL of the test substance (the finished product of Example 3 diluted with deionized water to a concentration of 0.1 mg / mL) and drop it into the conjunctival sac of one eye of the rabbit, passively close the eye for 1 s, do not rinse, and do not treat the other eye as a control. Check the animals' eyes at 1 h, 24 h, 48 h, 72 h, and on the 4th and 7th days after dropping the test substance. If no irritation reaction occurs within 72 h, the test can be terminated. If corneal involvement or other eye irritation effects are found and do not recover within 7 days, to determine the reversibility or irreversibility of the damage, the observation time needs to be extended, generally not exceeding 21 days, and observation reports for 7 days, 14 days, and 21 days should be provided.

[0145] Skin allergy test:

[0146] 1. Test animals: 30 Hartley guinea pigs (male), provided by Guangzhou Tianzhu Biotechnology Co., Ltd., production license number: SCXK(Yue)2024 - 0071; quality certificate number: 44838300000858. Adapt to the experimental animal room for at least 7 days before the test.

[0147] 2. Rearing environment: Normal environment, temperature 18°C - 29°C; relative humidity 30% - 70%, license number for use: SYXK(Yue)2022 - 0194.

[0148] 3. Feed source: Guangdong Provincial Center for Laboratory Animals in Medicine, production license number: Yue Sizheng(2024)05073, production date: 2025 - 05 - 16; feed certificate number: NO.2506060043 - 2.

[0149] 4. Positive substance: 2,4 - dinitrochlorobenzene (brand: Xiya Reagent; batch number: 20230614); pre - dissolved in acetone and then prepared with olive oil; induction concentration is 0.5%, excitation concentration is 0.2%.

[0150] 5. Test method:

[0151] 5.1 24 hours before the test, depilate the left side of the guinea pig's back, and the depilated area is about 4 cm 2 ~6 cm 2 ;

[0152] 5.2 Induction stage: Take 0.2 mL of the test substance and apply it to the 2 cm × 2 cm depilated test area on the left side of the back of the guinea pigs in the test group, then cover it with two layers of gauze and one layer of plastic film, and fix it with non - irritating adhesive tape. After 6 hours, wash off the test substance with warm water. Repeat the same method on the 7th day and the 14th day. The negative control group is not subjected to induction treatment.

[0153] 5.3 Excitation stage: 14 days after the last induction, take 0.2 mL of the test substance (the finished product of Example 3 diluted with deionized water to a concentration of 1 mg / mL) and apply it to the 2 cm × 2 cm depilated area on the right side of the back of the guinea pigs in the test group and the negative control group (shave the hair 24 hours before contact), then cover it with two layers of gauze and one layer of plastic film, and fix it with non - irritating adhesive tape. After 6 hours, wash off the test substance with warm water.

[0154] 5.4 Animal observation: Observe the skin reaction and score it 24 hours and 48 hours after the end of the excitation contact.

[0155] Skin phototoxicity test:

[0156] 1. Test animals: 6 ordinary - grade Hartley guinea pigs (half male and half female). Provided by Guangdong Provincial Center for Laboratory Animals in Medicine, production license number: SCXK(Yue)2023 - 0035; quality certificate number: NO.SCXK(Yue)2023 - 00352025003963. Adapt in the experimental animal house for at least 7 days before the test.

[0157] 2. Rearing environment: Ordinary environment, temperature 18°C - 29°C, relative humidity 30% - 70%, license number for use: SYXK(Guangdong)2022 - 0194.

[0158] 3. Feed source: Guangdong Provincial Medical Laboratory Animal Center, production license number: Guangdong Feed License(2024)05073, production date: 2025 - 07 - 29; feed certificate number: NO.2508080046.

[0159] 4. Instruments and equipment: Skin phototoxicity test detector (manufacturer: Tianjin Economic - Technological Development Area Hepu Industry and Trade Co., Ltd.; model: HOPE - MED8130B).

[0160] 5. Positive substance: 8 - Methoxypsoralen (brand:阿拉丁; batch number: 52317054; concentration: 0.05%), prepared with 95% ethanol.

[0161] 6. Test method:

[0162] 6.1 Preparation of test animals: 24 hours before the test, shave the hair on both sides of the spine on the back of the test animals without damaging the epidermis and with the skin of the test site intact. Prepare 4 hair - removal areas, each with a hair - removal area of about 2 cm × 2 cm, as shown in Figure 3 .

[0163] 6.2 Toxicity - application operation: Fix the animal, take 0.2 mL of the test substance and apply it to hair - removal areas 1 and 2 of the animal. After 30 minutes, cover the left side (hair - removal areas 1 and 3) with aluminum foil and fix it with tape, and irradiate the right side with UVA. The actual irradiation dose for the sample group is 10.0 J / cm 2 , and the UVB irradiation dose does not exceed 0.1 J / cm 2 .

[0164] 6.3 After the irradiation is completed, observe the skin reaction and score it at 1 h, 24 h, 48 h, and 72 h respectively.

[0165] See Figures 4-7 , the test results show that the finished product prepared by the present invention meets the relevant regulations of the "Cosmetics Safety and Technology Standards" (2015 Edition) and will not cause harm to human health under normal use conditions.

[0166] The above description is a detailed description of the preferred and feasible embodiments of the present invention, but the embodiments are not intended to limit the scope of the patent application of the present invention. Any equivalent changes or modifications made under the technical spirit disclosed by the present invention shall fall within the scope of the patent covered by the present invention.

Claims

1. A method for preparing tremella gum, characterized in that, The preparation method of the tremella gel includes the following steps: (1) The dried fruiting body of Tremella fuciformis is crushed and sieved to obtain Tremella fuciformis powder; (2) The tremella powder and the extraction solvent were mixed and then subjected to ultrasonic treatment to obtain a mixture; (3) Add an inorganic base to the mixture and stir to mix. While stirring, slowly add propylene oxide, keep the reaction at a constant temperature, add an acid to adjust the pH to 5-8, and dry to obtain the tremella gel. The water content of the extraction solvent is 15wt%-20wt%; The ratio of the tremella powder to the extraction solvent is 1g: 5-10mL; The ultrasonic treatment is performed by first ultrasonicating at a temperature of 40-50℃ and an ultrasonic frequency of 100-180W for 10-12 minutes, and then ultrasonicating at a temperature of 70-80℃ and an ultrasonic frequency of 200-300W for 3-5 minutes. The extraction solvent comprises betaine, polyethylene glycol, and lactic acid in a molar ratio of 1:(2-4):(0.4-0.8); Based on the mass of the tremella powder, the amount of propylene oxide is 30%-50%, and the amount of inorganic alkali is 10%-25%.

2. The method of claim 1, wherein the preparation of the tremella gum is characterized by, The inorganic base is any one of sodium bicarbonate, sodium carbonate, sodium hydroxide, potassium hydroxide, potassium carbonate, and potassium bicarbonate.

3. The method of claim 1, wherein the preparation of the tremella gum is characterized by, In step (3), the stirring speed is 200-400 r / min, the temperature is 45-55℃, and the time is 0.25-0.5 h. When adding propylene oxide, the stirring speed is 200-300 r / min, the temperature is 50-70℃, and the time is 1-2 h. The stirring speed for the heat preservation reaction is 50-150 r / min, the temperature is 50-70℃, and the time is 2-4 h.

4. The method of claim 1, wherein the preparation of the tremella gum is characterized by, The polyethylene glycol is polyethylene glycol 400.

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